RESUMO
BACKGROUND: Circular RNAs (circRNAs) have important regulatory functions in cancer, but the role of circRNAs in the tumor microenvironment (TME) remains unclear. Moreover, we also explore the effects of si-circRNAs loaded in nanoparticles as therapeutic agent for anti-tumor in vivo. METHODS: We conducted bioinformatics analysis, qRT-PCR, EdU assays, Transwell assays, co-culture system and multiple orthotopic xenograft models to investigate the expression and function of circRNAs. Additionally, PLGA-based nanoparticles loaded with si-circRNAs were used to evaluate the potential of nanotherapeutic strategy in anti-tumor response. RESULTS: We identified oncogene SERPINE2 derived circRNA, named as cSERPINE2, which was notably elevated in breast cancer and was closely related to poor clinical outcome. Functionally, tumor exosomal cSERPINE2 was shuttled to tumor associated macrophages (TAMs) and enhanced the secretion of Interleukin-6 (IL-6), leading to increased proliferation and invasion of breast cancer cells. Furthermore, IL-6 in turn increased the EIF4A3 and CCL2 levels within tumor cells in a positive feedback mechanism, further enhancing tumor cSERPINE2 biogenesis and promoting the recruitment of TAMs. More importantly, we developed a PLGA-based nanoparticle loaded with si-cSERPINE2, which effectively attenuated breast cancer progression in vivo. CONCLUSIONS: Our study illustrates a novel mechanism that tumor exosomal cSERPINE2 mediates a positive feedback loop between tumor cells and TAMs to promote cancer progression, which may serve as a promising nanotherapeutic strategy for the treatment of breast cancer.
Assuntos
Neoplasias da Mama , RNA Circular , Feminino , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Interleucina-6/metabolismo , Macrófagos/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Serpina E2/metabolismo , Serpina E2/farmacologia , Microambiente Tumoral , Macrófagos Associados a Tumor/metabolismo , AnimaisRESUMO
Cardiac fibrosis is one of the common pathological processes in many cardiovascular diseases characterized by excessive extracellular matrix deposition. SerpinE2 is a kind of protein that inhibits peptidase in extracellular matrix and up-regulated tremendously in mouse model of cardiac fibrosis induced by pressure-overloaded via transverse aortic constriction (TAC) surgery. However, its effect on cardiac fibroblasts (CFs), collagen secretion and the underlying mechanism remains unclear. In this study, DyLight® 488 green fluorescent dye or His-tagged proteins were used to label the exogenous serpinE2 protein. It was showed that extracellular serpinE2 translocated into CFs by low-density lipoprotein receptor-related protein 1 (LRP1) and urokinase plasminogen activator receptor (uPAR) of cell membrane through endocytosis. Knockdown of LRP1 or uPAR reduced the level of serpinE2 in CFs and down-regulated the collagen expression. Inhibition of the endocytosis of serpinE2 could inhibit ERK1/2 and ß-catenin signaling pathways and subsequently attenuated collagen secretion. Knockdown of serpinE2 attenuates cardiac fibrosis in TAC mouse. We conclude that serpinE2 could be translocated into cardiac fibroblasts due to endocytosis through directly interact with the membrane protein LRP1 and uPAR, and this process activated the ERK1/2, ß-catenin signaling pathways, consequently promoting collagen production.
Assuntos
beta Catenina , Camundongos , Animais , beta Catenina/metabolismo , Serpina E2/metabolismo , Serpina E2/farmacologia , Inibidores de Proteases/farmacologia , Sistema de Sinalização das MAP Quinases/genética , Fibrose , Transdução de Sinais/genética , Endocitose/genética , Colágeno/metabolismoRESUMO
Prostate cancer (PCa) is a common malignant cancer of the urinary system. Drug therapy, chemotherapy, and radical prostatectomy are the primary treatment methods, but drug resistance and postoperative recurrence often occur. Therefore, seeking novel anti-tumor compounds with high efficiency and low toxicity from natural products can produce a new tumor treatment method. Matijin-Su [N-(N-benzoyl-L-phenylalanyl)-O-acetyl-L-phenylalanol, MTS] is a phenylalanine dipeptide monomer compound that is isolated from the Chinese ethnic medicine Matijin (Dichondra repens Forst.). Its derivatives exhibit various pharmacological activities, especially anti-tumor. Among them, the novel MTS derivative HXL131 has a significant inhibitory effect against prostate tumor growth and metastasis. This study is designed to investigate the effects of HXL131 on the growth and metastasis of human PCa cell lines PC3 and its molecular mechanism through in vitro experiments combined with proteomics, molecular docking, and gene silencing. The in vitro results showed that HXL131 concentration dependently inhibited PC3 cell proliferation, induced apoptosis, arrested cell cycle at the G2/M phase, and inhibited cell migration capacity. A proteomic analysis and a Western blot showed that HXL131 up-regulated the expression of proliferation, apoptosis, cell cycle, and migration-related proteins CYR61, TIMP1, SOD2, IL6, SERPINE2, DUSP1, TNFSF9, OSMR, TNFRSF10D, and TNFRSF12A. Molecular docking, a cellular thermal shift assay (CETSA), and gene silencing showed that HXL131 had a strong binding affinity with DUSP1 and TNFSF9, which are important target genes for inhibiting the growth and metastasis of PC3 cells. This study demonstrates that HXL131 exhibited excellent anti-prostate cancer activity and inhibited the growth and metastasis of prostate cancer cells by regulating the expression of DUSP1 and TNFSF9.
Assuntos
Produtos Biológicos , Neoplasias da Próstata , Ligante 4-1BB , Apoptose , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Fosfatase 1 de Especificidade Dupla/genética , Humanos , Interleucina-6/farmacologia , Masculino , Simulação de Acoplamento Molecular , Fenilalanina/farmacologia , Neoplasias da Próstata/metabolismo , Proteômica , Serpina E2/farmacologiaRESUMO
OBJECTIVE: To explore the role of protease nexin-1 (PN-1) in Alzheimer's disease (AD) via the sonic hedgehog (SHH) pathway. METHODS: PN-1 lentiviral activation particles were injected into APP/PS1 transgenic AD and wild-type (WT) mice; these mice were subjected to the Morris water maze test, followed by ELISA, thioflavin S staining and NeuN-TUNEL dual staining. HT22 cells were induced with Aß1-42 and treated with PN-1 siRNA and/or cyclopamine (an SHH signaling inhibitor). The cells were then subjected to MTT and Annexin V-FITC/PI analyses. qRT-PCR and Western blotting were conducted to measure mRNA and protein expression. RESULTS: The escape latency of the APP/PS1 transgenic AD mice was extended with a decreased number of platform crossings; in addition, increased Aß deposits, Aß1-42 levels and hippocampal neuron apoptosis were observed in the brain tissues of AD mice. However, these changes were improved by PN-1 lentiviral activation particles. In addition, PN-1 overexpression inhibited the SHH pathway in AD mice. Moreover, PN-1 overexpression abolished the Aß1-42-induced activation of the SHH pathway in HT22 cells. In addition, Aß1-42 induction resulted in an increased apoptotic rate and decreased cell viability of HT22 cells; however, these effects were reversed by PN-1 or cyclopamine. Compared with that in the PN-1 siRNA + cyclopamine + Aß1-42 group, apoptosis of HT22 cells in the cyclopamine + Aß1-42 group was reduced and cell viability was improved. CONCLUSION: PN-1, by blocking SHH pathway, reduced apoptosis of hippocampal neurons to improve spatial learning and memory ability, thereby playing a protective role in AD.
Assuntos
Doença de Alzheimer/metabolismo , Apoptose/fisiologia , Proteínas Hedgehog/metabolismo , Serpina E2/metabolismo , Transdução de Sinais/fisiologia , Animais , Comportamento Animal/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Hipocampo , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Serpina E2/farmacologiaRESUMO
The serine proteases, tissue- and urokinase-type plasminogen activators (PLAT and PLAU) and their inhibitors SERPINE1/2 are regulators of plasminogen to plasmin conversion. They are widely expressed in ovarian tissues, including granulosa and cumulus cells, and their expression is regulated by gonadotropins. The aim of this work was to assess the effect of serine protease inhibitors (aprotinin and AEBSF) and SERPINE1/2 on FSH-induced cumulus cell expansion, the production of prostaglandin E2 (PGE2) and retention of hyaluronic acid (HA) in expanding cumulus. The serine protease activity proved to be essential for the production of PGE2 and also for the retention of HA; the inhibition of plasminogen activators by SERPINE1/2 had the same effect. Collectively, these data indicate that plasmin is required for proper function of expanding cumulus cells in vitro and presumably also in vivo in the pre-ovulatory follicles.
Assuntos
Células do Cúmulo/efeitos dos fármacos , Dinoprostona/metabolismo , Oócitos/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Inibidores de Serina Proteinase/farmacologia , Serpina E2/farmacologia , Animais , Aprotinina/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Ácido Hialurônico/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Sulfonas/farmacologia , SuínosRESUMO
SERPINE2 (serpin peptidase inhibitor, clade E, member 2), predominantly expressed in the seminal vesicle, can inhibit murine sperm capacitation, suggesting its role as a sperm decapacitation factor (DF). A characteristic of DF is its ability to reverse the capacitation process. Here, we investigated whether SERPINE2 can reversibly modulate sperm capacitation. Immunocytochemical staining revealed that SERPINE2 was bound onto both capacitated and uncapacitated sperm. It reversed the increase in BSA-induced sperm protein tyrosine phosphorylation levels. The effective dose and incubation time were found to be >0.1 mg/mL and >60 min, respectively. Calcium ion levels in the capacitated sperm were reduced to a level similar to that in uncapacitated sperm after 90 min of incubation with SERPINE2. In addition, the acrosome reaction of capacitated sperm was inhibited after 90 min of incubation with SERPINE2. Oviductal sperm was readily induced to undergo the acrosome reaction using the A23187 ionophore; however, the acrosome reaction was significantly reduced after incubation with SERPINE2 for 60 and 120 min. These findings suggested that SERPINE2 prevented as well as reversed sperm capacitation in vitro. It also prevented the acrosome reaction in in vivo-capacitated sperm isolated from the oviduct. Thus, SERPINE2 could reversibly modulate murine sperm capacitation.
Assuntos
Reação Acrossômica , Acrossomo/efeitos dos fármacos , Serpina E2/farmacologia , Acrossomo/metabolismo , Animais , Cálcio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Serpina E2/metabolismoRESUMO
PURPOSE: Protease nexin-1 (PN-1), a serpin encoded by the SERPINE2 gene, has serine protease inhibitory activity and neurotrophic properties in the brain. PN-1 inhibits retinal angiogenesis; however, PN-1's neurotrophic capacities in the retina have not yet been evaluated. Pigment epithelium-derived factor (PEDF) is a serpin that exhibits neurotrophic and antiangiogenic activities but lacks protease inhibitory properties. The aim of this study is to compare PN-1 and PEDF. METHODS: Sequence comparisons were performed using computer bioinformatics programs. Mouse and bovine eyes, human retina tissue, and ARPE-19 cells were used to prepare RNA and protein samples. Interphotoreceptor matrix lavage was obtained from bovine eyes. Gene expression and protein levels were evaluated with reverse-transcription PCR (RT-PCR) and western blotting, respectively. Recombinant human PN-1, a version of PN-1 referred to as PN-1[R346A] lacking serine protease inhibitory activity, and PEDF proteins were used, as well as synthetic peptides designed from PEDF and PN-1 sequences. Survival activity in serum-starved, rat-derived retinal precursor (R28) cells was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) cell death assays. Bcl2 levels were measured with RT-PCR. RESULTS: PN-1 is analogous in primary and tertiary structure to PEDF. A region in PN-1 shares homology with the neurotrophic active region of PEDF, a 17-residue region within alpha helix C. The native human retina, ARPE-19 cells, and murine RPE and retina expressed the gene for PN-1 (SERPINE2 and Serpine2 mRNA). The retina, ARPE-19 cell lysates, and bovine interphotoreceptor matrix contained PN-1 protein. The addition of PN-1, PN-1[R346A], or the 17mer peptide of PN-1 to serum-starved retina cells decreased the number of TUNEL-positive nuclei relative to the untreated cells, such as PEDF. PN-1, PN-1[R346A], and PN-1-17mer treatments increased the Bcl2 transcript levels in serum-starved cells, as seen with PEDF. CONCLUSIONS: PN-1 and PEDF share structural and functional features, and expression patterns in the retina. These serpins' mechanisms of action as cell survival factors are independent of serine protease inhibition. We have identified PN-1 as a novel factor for the retina that may play a neuroprotective role in vivo, and small peptides as relevant candidates for preventing retinal degeneration.
Assuntos
Proteínas do Olho/química , Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/farmacologia , Serpina E2/química , Serpina E2/farmacologia , Serpinas/química , Serpinas/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Bovinos , Linhagem Celular , Sobrevivência Celular , Citoproteção/efeitos dos fármacos , Olho/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Serpina E2/genética , Serpina E2/metabolismo , Homologia Estrutural de ProteínaRESUMO
Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis.
Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Serpina E2/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Células HL-60 , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Knockout , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Serpina E2/metabolismo , Serpina E2/farmacologia , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONALE: The antiangiogenic activity of rPAI-1(23), a truncated plasminogen activator inhibitor-1 (PAI-1) protein, induces vasa vasorum collapse and significantly reduces plaque area and plaque cholesterol in hypercholesterolemic low-density lipoprotein receptor-deficient/apolipoprotein B48-deficient mice. OBJECTIVE: The objective of this study was to examine rPAI-1(23)-stimulated mechanisms that cause vasa vasorum collapse. METHODS AND RESULTS: The rPAI-1(23) protein opposed PAI-1 antiproteolytic function by stimulating a 1.6-fold increase in plasmin activity compared with the saline-treated counterpart. The increased proteolytic activity corresponded to increased activity of matrix metalloproteinase-3 and degradation of fibrin(ogen), nidogen, and perlecan in the adventitia of descending aortas. PAI-1 activity was reduced by 48% in response to rPAI-1(23); however, PAI-1 protein expression levels were similar in the rPAI-1(23)- and saline-treated hypercholesterolemic mice. Coimmunoprecipitation assays demonstrated a novel PAI-1-plasminogen complex in protein from the descending aorta of rPAI-1(23)- and saline-treated mice, but complexed PAI-1 was 1.6-fold greater in rPAI-1(23)-treated mice. Biochemical analyses demonstrated that rPAI-1(23) and PAI-1 binding interactions with plasminogen increased plasmin activity and reduced PAI-1 antiproteolytic activity. CONCLUSIONS: We conclude that rPAI-1(23) causes regression or collapse of adventitial vasa vasorum in hypercholesterolemic mice by stimulating an increase in plasmin activity. The rPAI-1(23)-enhanced plasmin activity was achieved through a novel mechanism by which rPAI-1(23) and PAI-1 bound plasminogen in a cooperative manner to increase plasmin activity and reduce PAI-1 activity.
Assuntos
Inibidores da Angiogênese/farmacologia , Fibrinolisina/metabolismo , Hipercolesterolemia/metabolismo , Plasminogênio/metabolismo , Serpina E2/farmacologia , Vasa Vasorum/metabolismo , Animais , Fibrinolisina/genética , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Camundongos , Camundongos Knockout , Plasminogênio/genética , Vasa Vasorum/patologiaRESUMO
Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1-20 nM) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor L-NAME (N(D)-nitro-L-arginine methyl ester). Two-chain uPA (1-20 nM) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser(1177) in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of ß-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPA-induced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA- and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPA-based approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.
Assuntos
Barreira Alveolocapilar/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Mucosa Respiratória/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Barreira Alveolocapilar/patologia , Permeabilidade Capilar/genética , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Edema Pulmonar/genética , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Mucosa Respiratória/patologia , Serpina E2/genética , Serpina E2/metabolismo , Serpina E2/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
SERPINE2, one of the potent serine protease inhibitors that modulates the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the present study, we purified SERPINE2 from mouse seminal vesicle secretion (SVS), using liquid chromatography and identified it by liquid chromatography/tandem mass spectrometry, and it showed potent inhibitory activity against the urokinase-type plasminogen activator. SERPINE2 was expressed predominantly in seminal vesicles among murine male reproductive tissues. It was immunolocalized to the SVS and mucosal epithelium of the seminal vesicle, epididymis, coagulating gland, and vas deferens. In the testes, SERPINE2 was immunostained in spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa. SERPINE2 was also detected on the acrosomal cap of testicular and epididymal sperm and was suggested to be an intrinsic sperm surface protein. The purified SERPINE2 protein could bind to epididymal sperm. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa. Nevertheless, SERPINE2 was detected predominantly on uncapacitated sperm, indicating that SERPINE2 is lost before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that the SERPINE2 protein may play a role as a sperm decapacitation factor.